Project Summary Accurate DNA replication is essential for preserving genomic stability and protecting against carcinogenesis. Obstacles to DNA replication, such as DNA adducts, secondary DNA structures, or nucleotide depletion block the progression of DNA polymerases and thus arrest replication forks, which can lead to fork collapse and DNA breaks. As this is a source for point mutations and structural variations, understanding replication stress is important for both cancer prevention and treatment. Stalled replication forks can be reversed, a process which stabilizes stalled forks, but also exposes them to nucleolytical degradation, unless protected through loading of RAD51 by the FA-BRCA pathway. PCNA is an essential replication fork component, which upon exposure to DNA damage is ubiquitinated at lysine (K) 164. This event controls two mechanisms of DNA lesion bypass, translesion synthesis and template switching. While traditionally PCNA mono-ubiquitination was considered a DNA damage-induced event, recent work by our laboratories and others suggested a more broad impact of PCNA ubiquitination during replication. To mechanistically address this, we employed the CRISPR/Cas9 genome editing technology to introduce the K164R mutation in the endogenous PCNA alleles in HEK293T, RPE1, and HCT116 cells. Preliminary characterization of these cells revealed unexpected genomic instability features, including telomere erosion, replication fork degradation, and gross chromosomal rearrangements. Based on these preliminary results, we propose that PCNA ubiquitination has previously unrecognized roles in regulating genomic stability. We will address this in three specific aims: Aim 1 will investigate the role of PCNA modification at K164 in telomere maintenance. Aim 2 will investigate the role of K164-modified PCNA in suppressing replication fork degradation. Aim 3 will elucidate the functional impact of PCNA modification at K164 on genomic rearrangements. Our work is poised to uncover novel cellular mechanisms regulated by PCNA modification at K164, and will have a significant impact on our understanding of genome integrity.